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Histopathology of a granulosis in the larva of the fall webworm, Hyphantria cunea
JOURNAL
OF
IKVEHTEBRATE
PATHOLOGY
Histopathology Fall HITOSHI
16,
71-79
(1970)
of a Granulosis in the Larva Webworm, Hyphuntria cunea WATANABEI
Receiced
AND
MASAIXIKO
Nocemher
14,
of the
KOBAYASHI
1969
Light-autoradiography, light microscope, and electron microscope observations of the larval fat body of the fall webworm, Hyplaantria CU~~CU, infected with a granlllosis virus revealed that a mitotic cell proliferation and nuclear hypertrophy of the fat-body cells occurred at an early stage of infection, and sllbsequently, a stained network developed in the entire area of the enormously hypertrophied nuclei. A number of virus rods appeared to protrude from the strands of very dense material of the network into the spaces between the strands. where the virus rods were env-eloped with the capsule protein. Light-autoradiography preparations revealed that areas of active synthesis of Dh’A, RNA, and protein were restricted to the nucleus at an early stage of infection, and at a later stage, the active synthesis was restricted arolmd the strands of network. These results suggested that, although the development of grannlosis virus begins in the cell nucleus and continues after early rupture oi the nuclear membrane in both the nuclear and cytoplasmic areas, the material usrd in the formation of the granulosis virus is probably of nuclear origin. In the fat body infected simultaneously with granulosis and ntIcleopolvhedrosis viruses, no cell with both virus types of infection \vas detected with the iight and electron nlicroscopes.
IXlTHODUCTION
A granulosis of the fall webworm, Hycunea, was first reported bv Schmidt and Philips ( 1958). Although this granulosis has been studied with regards to its etiology, signs and svmptoms, and epidemiology ( KovaCevib, i958; Weizer, 1958; Hukuhara and Hashimoto, 1966; Hukuhara et al., 1969), there is no detailed histopathological studv of the grallulosis in H. cunea. Accordingly; we have conducted such a studv of the granulosis in H. cunea larva using light and electron microscope techniques and light autoradiography. The results presented here emphasize the following points: the sequence of cytopath-
phantriu
1 Present address: Division of Entomology, Hilcrard Hall 333, University of California, Berkeley, &Mhnia 94720.
71
ological events in the infected fat-bode cell; the changes of nucleic acid and protein svnthesis activities in the infected ccl1 during the course of the granulosis; aiid histopathologv of a fat body infected simultaneously with granulosis and m&opol~~hedrosis viruses. MATEHIALS
AND
METHODS
Materials. Larvae of the fall wcbworn I were obtained from field-collected eggs cmcl reared in the laboratorv on mulbcrrv Icaves. The* rarlv ijtll-instar larvae wercaallowed to feed for. 24 hr on leaves treated with ;I heavv suspension of the granulosis virus to cause 100% infection. To study the simultaneous infection with granulosis and n\~cleopolvhedrosis viruses, other larvae wer(a f0rcc.d fct? ;in inocuhim containirq it inix-
72
WATANABE
AND
ture of granulosis and polyhedrosis viruses. Samples for light and electron microscope studies were taken at daily intervals after inoculation. Light microscopy. Larval body parts containing fat body were fixed in Carnoy’s fluid or in Bouin-Duboscq solution, dehydrated in an alcohol series, and embedded in paraffin. Sections were cut at S-7 u and stained with Harris’s hematoxylin-eosin or Giemsa’s stain after hydrolysis (10 min in 1.0 N HCl at 6O’C). For some sections, the azan staining techniques developed by Hamm ( 1966) were used. Light autoradiography. For the determination of DNA, RNA, and protein synthesis activities in the infected fat body during the course of granulosis, autoradiographs of the tissues were prepared by using 3H-thymidine, 3H-uridine, and 3H-tyrosine (Watanabe, 1967, 196S). Electron microscopy. Fat body was fixed in 2.5% glutaraldehyde in 0.05 M sodium cacodylate buffer, postfixed in 1% osmium tetroxide in Palade’s phosphate buffer, dehydrated in an ethanol series, and embedded in Epon. Sections were cut with an LKB ultratome, double stained in uranyl acetate and lead citrate, and examined with a JEM5Y electron microscope. RESULTS
AND
DISCUSSION
Characteristics of the disease. Larvae of H. cunea, when affected by the granulosis, lost their appetite and later ceased feeding. The infected larvae became more or less swollen, and the ventral portion of the abdomen turned yellowish in color at a late stage of infection. The blood from the heavily diseased larvae was milky and contained large numbers of capsules and remnants of disintegrated fat body. Before death, the diseased larvae often hung by their prolegs from the wall of the rearing container, showing signs similar to that of
KOBAYASHI
larvae with nucleopolyhedrosis but with integument that remained relatively firm. The period of lethal granulosis infection varied considerably with the different larval stages, the rearing temperature, and the dosage of virus inoculum but most of the mortality occurred in 10 to 20 days after infection. Light microscope histopathology. In larvae infected with granulosis virus, the fat body was the only tissue affected. Preparations of the fat body 1 or 2 days after infection revealed mitotic cell proliferations and hypertrophied nuclei (Fig. 1) . This initial sign of infection also has been observed in the granuloses of other insect species (Martignoni, 1957; Wittig, 1959a; Hamm, 1968 ) . As the nucleus continued to hypertrophy, the fat globules in the cytoplasm became voluminous and increased the size of the entire cell (Fig. 2). Subsequently, the nuclear material in the infected cell seemed to break up and condense into irregularly shaped masses. At the same time, some round structures, which did not stain and contained granulated chromatin remnants, were frequently observed in the degenerating nucleus (Fig. 3). These round structures fused and formed a continuous ring of degenerating nuclear material lying just within the periphery of the nuclear membrane. The center space of the ring was filled with chromatin remnants (Fig. 4) that gradually expanded until the whole nucleus became a vague granuIar mass (Fig. 5). At a more advanced stage of the disease, an intensely stained network developed throughout the enormously hypertrophied nucleus that occupied a large portion of the cell (Fig. 6). By this time, the nuclear membrane had ruptured, and the nuclear material mingled with the cytoplasmic components. The development of the network was the most characteristic feature of the
HISTOPATHOLOCY
disease and had already been observed in other granuloses (Hughes and Thompson, 1951; Wittig and Franz, 1957; Bird, 1958; Wittig, 1959a, b; Huger, 1960). Finally, in the fat body of moribund larvae, the network disintegrated and was followed
OF
A
GRANULOSIS
shortly brane.
bv the rupture
r. 1, 3
of the cell men)-
Light microscope autoradiography. Autoradiographs of the infected fat body treat& with “H-thymidine revealed that a large amount of thr lahrl was incorporated into
Frcs. 1-6. Histopathological changes in fat-body cells of Hyphantria cunea larvae during the course of granulosis. Photomicrographs of sections stained with Harris’s hematoxylin-eosin. Fig. 1. Two days after infection showing proliferation of cells. X 120. Fig. 2. Three days after infection showing increase in size of entire cell and fat globules in the cytoplasm. X680. Figs. 3 and 4. Degeneration of nucleus containing granulated chromatin remnants at 4 days after infection. X680. Fig. 5. Six days after infection showing nuclear content altered into a vague granular mass. X 680. Fig. 6. Eight d:rys after infection showing development of network in the virogenic stroma X680.
74
WATANABE
AND
the nuclei 1 or 2 days after inoculation with the virus. This suggested that an active synthesis of DNA was occurring in the nuclei (Figs. 7 and 8). The newly synthesized DNA at the very earlv stage of infection was probably not onlv -viral DNA, but also host DNA associated With the cell proliferation induced by the virus. As the infected nuclei hypertrophied, most of the radioactivity appeared in the nuclear area but not in the cytoplasm (Figs. 9 and 10). At a later stage of infection, when a network developed throughout the enormously hypertrophied nuclei, the incorporation of the radioactive thymidine into the nuclei was greatly reduced, but most of the radioactivity appeared along the intensively stained strands of network, but some of theradioactivity was scattered over the disintegrated cytoplasmic area (Fig. 10). These autoradiographs clearly showed that
KOBAYASHI
most of the viral DNA was actively synthesized in the infected nucleus and the synthesis continued as the nucleus increased in size up to the time of network formation. In the autoradiographs obtained with ;‘H-uridinc, most of the radioactive uridinc during the course of the granulosis was incorporated into the nucleus, but some was distributed uniformly in the cytoplasm. The uptake of the radioactive uridine into the nucleus generally occurred in the early stage of infection, and as the disease advanced and the nucleus increased in size, the localization of the “H-uridine differed from that of “H-thymidine. In the infected nucleus, which includes some round structures surrounded with nuclear material, the uptake of the radioactive uridine occurred actively around the periphery of the round structures (Fig. 11). However, in the infected nucleus whose nuclear material had
FIGS. 7-10. Autoradiographs with :jH-thymidine demonstrating body cells of Hyplaantria cunea larvae infected with a granulosis X680. Sections of fat tissues 2 (Figs. 7 and 8), 6 (Fig. 9), and
DNA synthesis activities in fatvirus. Sections stained with Giemsa. 9 days (Fig. 10) after infection.
HISTOPATHOLOGY
OF
A GKASULOSIS
r-4 / ‘J
Fxs. 1 I-16. Arltoladiographs with :sH-midine demonstrating HSA synthesis activities in fat-hod\, cells of Hyphantrin wwxz larvae infected with a granulosis virns. Sections of fat tissllrs 1 ( Fii. Figs. l-1-16. Autoradiographs with :kH-t! 11), 8 (Fig. 12), and 9 days (Fig. 13) after infection. rosine demonstrating protein synthesis activities in fat-bodv cells of H. cutwa larvae infected with a granulosis virus. Sections stained with Ciemsa. Sections of fat tissues d (Fig. 14 ), 6 (Fig. I,5 ). :~uld 9 days (Fig. I(i) after infection.
76
WATANABEAND KOBAYASHI
condensed into irregular masses, the radioactive uridine was incorporated more or less uniformly throughout the whole nucleus (Fig. 12). In the infected nucleus with vague granular mass just prior to the formation of the network, the uptake of radioactive uridine occurred mainly around the granulated nuclear material. After the network had developed, the uptake of radioactive uridine was reduced, but some of the radioactivity appeared over the strands of the network (Fig. 13). Autoradiographs of infected cells treated with 3H-tyrosine during the course of granulosis indicated that the incorporation pattern and the amount of uptake of “H-tyro-’ sine were similar to those of 3H-uridine (Figs. 14-16). This suggested that protein synthesis in the infected cell during the course of the granulosis progressed in close association with RNA synthesis. Protein synthesis was pronounced in the whole nucleus up to the time of network formation, and thereafter the main part of the protein synthesis was restricted generally to the strands of network. Electron microscopy. At present the hypothesis is that the development of granulosis virus begins in the cell nucleus and continues, after an early rupture of the nuclear membrane, in both the nucleus and cytoplasmic areas (Huger and Krieg, 1961; Bird, 1963). Our electron microscope observations and autoradiographs presented herein support this hypothesis, i.e., that the granulosis virus is probably of nuclear origin. Electron micrographs of the infected fat body at a stage when the virogenic stroma exhibited a network revealed that a number
of virus rods appeared to protrude from strands of very dense material of the network into the spaces between the strands, where the rods were enveloped with a double membrane (Figs. 17 and 18). The mrclear membrane was completely destroyed by this time. Some long filaments of the‘ same thickness and density as virus rods were observed during the development of the virus ( Steinhaus et al., 1949; Smith aud Brown, 1965). Around the periphery of the strands, the virus particles were occluded in capsule protein and the capsules accumulated in the spaces between the strands. The occlusion of the virus particle by the capsule protein began mostly at one end of the virus rod as reported by Hughes (1952) and Bird (1964) and rarely began at both ends at the same time as observccl by Arnott and Smith (1968). The virus particles contained in the capsule were rod-shaped and varied from 5060 nq4 in diameter and from 280-350 rn!k in length. The capsules varied in size from 220-340 rnp in diameter and from 430600 rnp in length. The macromolecular lattice of the capsule protein was observed in thin sections. Double infection of granulosis and nucleopolyhedrosis viruses. Several insect species have been found infected simultaueouslv with both granulosis and nucleopolyhedrosis viruses (Tanada, 1953, 1956; Steinhaus, 1957; Bird, 1959). In H. cunea, mixed infection by granulosis and nucleopolyhedrosis viruses was obtained by feeding the larvae the mulberry leaves smeared with a mixed inoculum of the two viruses. The light microscope sections of the fat body with mixed virus infections showed
FIG. 17. Section through a fat-body cell of a larva of Hyplzat~tria cunea infected with a granulosis virus. Portion of virogenic stroma showing an abundance of capsules in spaces between the network strands and a number of virus rods scattered on the strands. X6,500. FIG. 18. Section through a fat-body cell of a larva of Hyphantria cuneu infected with a granulosis virus. Portion of virogenic stroma showing protrusion of virus rods from the strands of network and the development of the capsule around the virus rod. X 17,000.
HISTOPATHOLOGY
OY
A C;HA h Ii L,c his
.&i
78
WATANABE
AND
POBAYASHI
FIG. 19. Section through a fat body of a larva of Hyphtria curlea infected simultaneously with a nucleopolyhedrosis and a granulosis virus. The left cell is infected with a nucleopolyhedrosis virus and the right cell with a granulosis virus. Nucleopolyhedrosis virus ( NV), polyhedra (P), nuclear membrane (NM), cell membrane ((%I), granulosis virus rod (V), and capsule (C). X6,000.
that no one cell was infected simultaneoushby both viruses. This was aIso the case with the mixed virus infections in the larvae of Chorktoneura fumiferana and Trichoplusia ni according to Bird (1959) and Lowe and Paschke (1968). Although we have made numerous attempts with the electron microscope to find a cell with mixed virus infections, we were unsuccessful. However, WC’ did observe adjacent cells infected by different viruses (Fig. 19).
ACKWWLUXXENTS
Appreciation is expressed to Professor Y. Tanada of University of California for reviewing the manuscript and offering suggestions for its improvement.
REFEREAT%
H. J., ANll ShfITH, K. M. 1968. An ultrastructural study of the development of granulosis virus in the cells of the moth, Plodia interpunctellu ( Hbn. ). J. Ultrastruct. Res., 21, 251-268. Brim, F. T. 1958. Histopathologyof granulosis AHNOTT,
IIISTOPATHOL0C.T
viruses
in insects.
272. BIRD, F. T.
1959.
Cm.
J.
Microbial.,
Polyhedrosis
OF
Pathol.,
wrtebr.
8,
and granulosis
125-126.
J. J. 1968. Comparative of a granulosis and a nuclear Spocloptcru
frcrgipwlo.
10, 320-326. HKJcEx, A. 1960. \verkes bei der n~rrrimma ( Hiib. h~uttlr~isscrlsC/~~ft~i~,
iiber
1.
histopathology polyhedrosis
Inwrtehr.
die Natur
Cramdose van ) ( Lrpidoptera, 47,
of
Pdol.,
des FadenChoristoneuru
Tortricidae
).
3358-359.
HUGEN, A., A.\D I&EC, A. 1961. Electron microscope investigations on the virogenesis of the granulosis of Choristoncuru murinana ( Hiillner), J. Insrc? Pathl., 3, 183-196. HUGHES, K. M. 1952. Development of the inclusion bodies of a gl-anulosis virlls. J. Bacteriol.,
64,
37ii380.
AND T~>MPSOS, C. C. 1951. 11 of the omnivorous looper, Subt&Xles cdxmtn Clleni>r. J. Infcc. Dis., 89, 173-179. HUKUHAIK T., ANI) HASEIIMOTO, Y. 1966. Virus diseases of H!yphuntrk c~meu l)rl1ry. Jap. J. A/$. El~tonloz.Zool., 10, l-49-155. HUKUHAHA, 'I‘., i21%cc:a, H., ASD KOBAYASHI, hl. 1969. On the gran~Ilosis of Hyphantriu mnm L)rur\,. lap. J. A~$. Etltouwl. Z&l., 13, l-4. KoVAi:EVI& Z. 1958. Pathogene Mikroorganisnwn als Begleiter und hfortalitii:sfaktoren des amerikanischrn Webewaren, Hyplaantria cuffeci I>n1l-!-. Amz. Sch:rtllinjiskfrncle, 31, 14% 150. LOWE, R. E., ASI) PASCHKE, 1. L). 1968. Pathology of a double viral infection of Trichophia Iii. J. Illaeffelw. Putllol., 12, 438-443. ~~AWIC:SONI, XI. E. 1957. Contributo alla conoscmza di lma granulosi di Encosma griseurlc~ ( Ffiihnt~r) (Tortricidw, Lrpidoptera) quak
ljCTr,HEb,
K.
gramdoris
hf.,
70
4, 267-
virl~ses causing single and double infections in tile spruce budwom, Choristor~erm fcmif~ rcmci Clemens. J. fr1accf PUG&., 1, 406130. AIHD, F. T. 1963. On the development of granulosis viruses. J. Illsect Pnthol., 5, 368-376. HIHI). F. T. 196-l. On the development of insect polyhedrosis and grannlosis virus particles. Can. I. Itficwl~ioz., 10, 49-52. HAMhI, J. J. 1966. A modified azan staining technique for inclusion body viruses. J. InHAMM,
.4 (X4NIXOSIS
SWI'H.
h.
hf.,
AS,)
HIKWX,
H.
XI.
1965.
.\
study ot the long rods associatrd with irwc.t grannloses, ITirology, 27, 512-519. STEIKIMU. LC. :\. 19.57. Xetv records 01’ inswlvirlls diseases. Hilgartl!a, 26, 417~-130. STEISIIAUS. E. A., HLXEIES, h;. &I., ANI) \VASSEI~, H. B. 1949. Demonstration of the granlllosih \.irrls of the varigated cutworm. J. Bnc,tc~riol.. 57, 219-224. TASAIIA, B. 1953. Description and characteristics of a granlllosis virlls of the import& ~~blqewtrrm. Z’rw. Huwuii. Etltomol. Soc, , 15, 23,%260. ‘rAN,\I)A, Y. 1956. Some factors afl‘rcting tIlti s~isceptibilitv of the armyworm to virlls infwtion. J. Eco;~. 14:tltwlo/., 49. 52-57. \tT.4TANA13E, H. 1967. Site of viral lKi.~\ syntlrvsis \r?thin Inidgrlt cells of silk\vonn, Bomhr/x tJlOI’i. infected \vith Cvt~)pli~sn~iC-l~~)lvl~edrosi~ virws.
.l. llicertchr.
PfALOl.,
9, .18&&7.
Il. 1968. Light radioalltograpl)ic, atrldy of protein synthesis in the midgIlt epithelilnn ot the silkworm, Bodxyx nmi. infected \vith a ~~toplasmic-l)t)l~l~edrosis \+ll<. J. hccrtehr. I-‘c~tho/., 11, 310-31Fj. \~~EIZEH, 1. 19.58. Report on rewarcll work trf the laboratorv of insect pathology 1957 and 1955. Czwll. A&. Sci., Inst. Biol., l--&8. w1rm, (:. 1959a. I!ntersnchungen iiber dt.11 \‘rrlallf drr (:ranulose bri Ranpen \ 011 Cl~ori.rtonc,rrw r)turinurru. i ffiib. ) ( Lepidopt., ‘~‘ortricidar ). .Arcll. Cksant~~ \‘irrrsfor.w+ .. 9,
WATANABE:,
365-39,5.
(:. 19591). Ein Beitrag znr Histopathtrlogie dcr Kapsrlvirose \un Chori.~to~icwa m rrriuwu~ ( Hi&. ) ( Lrpidopt., Tortricidae ). Pror
~ITTIC:,
lnt. 898.
Co~rgr.
Crop.
Prot.
lth
H.
N57.
1. 89,T-
OF
IKVEHTEBRATE
PATHOLOGY
Histopathology Fall HITOSHI
16,
71-79
(1970)
of a Granulosis in the Larva Webworm, Hyphuntria cunea WATANABEI
Receiced
AND
MASAIXIKO
Nocemher
14,
of the
KOBAYASHI
1969
Light-autoradiography, light microscope, and electron microscope observations of the larval fat body of the fall webworm, Hyplaantria CU~~CU, infected with a granlllosis virus revealed that a mitotic cell proliferation and nuclear hypertrophy of the fat-body cells occurred at an early stage of infection, and sllbsequently, a stained network developed in the entire area of the enormously hypertrophied nuclei. A number of virus rods appeared to protrude from the strands of very dense material of the network into the spaces between the strands. where the virus rods were env-eloped with the capsule protein. Light-autoradiography preparations revealed that areas of active synthesis of Dh’A, RNA, and protein were restricted to the nucleus at an early stage of infection, and at a later stage, the active synthesis was restricted arolmd the strands of network. These results suggested that, although the development of grannlosis virus begins in the cell nucleus and continues after early rupture oi the nuclear membrane in both the nuclear and cytoplasmic areas, the material usrd in the formation of the granulosis virus is probably of nuclear origin. In the fat body infected simultaneously with granulosis and ntIcleopolvhedrosis viruses, no cell with both virus types of infection \vas detected with the iight and electron nlicroscopes.
IXlTHODUCTION
A granulosis of the fall webworm, Hycunea, was first reported bv Schmidt and Philips ( 1958). Although this granulosis has been studied with regards to its etiology, signs and svmptoms, and epidemiology ( KovaCevib, i958; Weizer, 1958; Hukuhara and Hashimoto, 1966; Hukuhara et al., 1969), there is no detailed histopathological studv of the grallulosis in H. cunea. Accordingly; we have conducted such a studv of the granulosis in H. cunea larva using light and electron microscope techniques and light autoradiography. The results presented here emphasize the following points: the sequence of cytopath-
phantriu
1 Present address: Division of Entomology, Hilcrard Hall 333, University of California, Berkeley, &Mhnia 94720.
71
ological events in the infected fat-bode cell; the changes of nucleic acid and protein svnthesis activities in the infected ccl1 during the course of the granulosis; aiid histopathologv of a fat body infected simultaneously with granulosis and m&opol~~hedrosis viruses. MATEHIALS
AND
METHODS
Materials. Larvae of the fall wcbworn I were obtained from field-collected eggs cmcl reared in the laboratorv on mulbcrrv Icaves. The* rarlv ijtll-instar larvae wercaallowed to feed for. 24 hr on leaves treated with ;I heavv suspension of the granulosis virus to cause 100% infection. To study the simultaneous infection with granulosis and n\~cleopolvhedrosis viruses, other larvae wer(a f0rcc.d fct? ;in inocuhim containirq it inix-
72
WATANABE
AND
ture of granulosis and polyhedrosis viruses. Samples for light and electron microscope studies were taken at daily intervals after inoculation. Light microscopy. Larval body parts containing fat body were fixed in Carnoy’s fluid or in Bouin-Duboscq solution, dehydrated in an alcohol series, and embedded in paraffin. Sections were cut at S-7 u and stained with Harris’s hematoxylin-eosin or Giemsa’s stain after hydrolysis (10 min in 1.0 N HCl at 6O’C). For some sections, the azan staining techniques developed by Hamm ( 1966) were used. Light autoradiography. For the determination of DNA, RNA, and protein synthesis activities in the infected fat body during the course of granulosis, autoradiographs of the tissues were prepared by using 3H-thymidine, 3H-uridine, and 3H-tyrosine (Watanabe, 1967, 196S). Electron microscopy. Fat body was fixed in 2.5% glutaraldehyde in 0.05 M sodium cacodylate buffer, postfixed in 1% osmium tetroxide in Palade’s phosphate buffer, dehydrated in an ethanol series, and embedded in Epon. Sections were cut with an LKB ultratome, double stained in uranyl acetate and lead citrate, and examined with a JEM5Y electron microscope. RESULTS
AND
DISCUSSION
Characteristics of the disease. Larvae of H. cunea, when affected by the granulosis, lost their appetite and later ceased feeding. The infected larvae became more or less swollen, and the ventral portion of the abdomen turned yellowish in color at a late stage of infection. The blood from the heavily diseased larvae was milky and contained large numbers of capsules and remnants of disintegrated fat body. Before death, the diseased larvae often hung by their prolegs from the wall of the rearing container, showing signs similar to that of
KOBAYASHI
larvae with nucleopolyhedrosis but with integument that remained relatively firm. The period of lethal granulosis infection varied considerably with the different larval stages, the rearing temperature, and the dosage of virus inoculum but most of the mortality occurred in 10 to 20 days after infection. Light microscope histopathology. In larvae infected with granulosis virus, the fat body was the only tissue affected. Preparations of the fat body 1 or 2 days after infection revealed mitotic cell proliferations and hypertrophied nuclei (Fig. 1) . This initial sign of infection also has been observed in the granuloses of other insect species (Martignoni, 1957; Wittig, 1959a; Hamm, 1968 ) . As the nucleus continued to hypertrophy, the fat globules in the cytoplasm became voluminous and increased the size of the entire cell (Fig. 2). Subsequently, the nuclear material in the infected cell seemed to break up and condense into irregularly shaped masses. At the same time, some round structures, which did not stain and contained granulated chromatin remnants, were frequently observed in the degenerating nucleus (Fig. 3). These round structures fused and formed a continuous ring of degenerating nuclear material lying just within the periphery of the nuclear membrane. The center space of the ring was filled with chromatin remnants (Fig. 4) that gradually expanded until the whole nucleus became a vague granuIar mass (Fig. 5). At a more advanced stage of the disease, an intensely stained network developed throughout the enormously hypertrophied nucleus that occupied a large portion of the cell (Fig. 6). By this time, the nuclear membrane had ruptured, and the nuclear material mingled with the cytoplasmic components. The development of the network was the most characteristic feature of the
HISTOPATHOLOCY
disease and had already been observed in other granuloses (Hughes and Thompson, 1951; Wittig and Franz, 1957; Bird, 1958; Wittig, 1959a, b; Huger, 1960). Finally, in the fat body of moribund larvae, the network disintegrated and was followed
OF
A
GRANULOSIS
shortly brane.
bv the rupture
r. 1, 3
of the cell men)-
Light microscope autoradiography. Autoradiographs of the infected fat body treat& with “H-thymidine revealed that a large amount of thr lahrl was incorporated into
Frcs. 1-6. Histopathological changes in fat-body cells of Hyphantria cunea larvae during the course of granulosis. Photomicrographs of sections stained with Harris’s hematoxylin-eosin. Fig. 1. Two days after infection showing proliferation of cells. X 120. Fig. 2. Three days after infection showing increase in size of entire cell and fat globules in the cytoplasm. X680. Figs. 3 and 4. Degeneration of nucleus containing granulated chromatin remnants at 4 days after infection. X680. Fig. 5. Six days after infection showing nuclear content altered into a vague granular mass. X 680. Fig. 6. Eight d:rys after infection showing development of network in the virogenic stroma X680.
74
WATANABE
AND
the nuclei 1 or 2 days after inoculation with the virus. This suggested that an active synthesis of DNA was occurring in the nuclei (Figs. 7 and 8). The newly synthesized DNA at the very earlv stage of infection was probably not onlv -viral DNA, but also host DNA associated With the cell proliferation induced by the virus. As the infected nuclei hypertrophied, most of the radioactivity appeared in the nuclear area but not in the cytoplasm (Figs. 9 and 10). At a later stage of infection, when a network developed throughout the enormously hypertrophied nuclei, the incorporation of the radioactive thymidine into the nuclei was greatly reduced, but most of the radioactivity appeared along the intensively stained strands of network, but some of theradioactivity was scattered over the disintegrated cytoplasmic area (Fig. 10). These autoradiographs clearly showed that
KOBAYASHI
most of the viral DNA was actively synthesized in the infected nucleus and the synthesis continued as the nucleus increased in size up to the time of network formation. In the autoradiographs obtained with ;‘H-uridinc, most of the radioactive uridinc during the course of the granulosis was incorporated into the nucleus, but some was distributed uniformly in the cytoplasm. The uptake of the radioactive uridine into the nucleus generally occurred in the early stage of infection, and as the disease advanced and the nucleus increased in size, the localization of the “H-uridine differed from that of “H-thymidine. In the infected nucleus, which includes some round structures surrounded with nuclear material, the uptake of the radioactive uridine occurred actively around the periphery of the round structures (Fig. 11). However, in the infected nucleus whose nuclear material had
FIGS. 7-10. Autoradiographs with :jH-thymidine demonstrating body cells of Hyplaantria cunea larvae infected with a granulosis X680. Sections of fat tissues 2 (Figs. 7 and 8), 6 (Fig. 9), and
DNA synthesis activities in fatvirus. Sections stained with Giemsa. 9 days (Fig. 10) after infection.
HISTOPATHOLOGY
OF
A GKASULOSIS
r-4 / ‘J
Fxs. 1 I-16. Arltoladiographs with :sH-midine demonstrating HSA synthesis activities in fat-hod\, cells of Hyphantrin wwxz larvae infected with a granulosis virns. Sections of fat tissllrs 1 ( Fii. Figs. l-1-16. Autoradiographs with :kH-t! 11), 8 (Fig. 12), and 9 days (Fig. 13) after infection. rosine demonstrating protein synthesis activities in fat-bodv cells of H. cutwa larvae infected with a granulosis virus. Sections stained with Ciemsa. Sections of fat tissues d (Fig. 14 ), 6 (Fig. I,5 ). :~uld 9 days (Fig. I(i) after infection.
76
WATANABEAND KOBAYASHI
condensed into irregular masses, the radioactive uridine was incorporated more or less uniformly throughout the whole nucleus (Fig. 12). In the infected nucleus with vague granular mass just prior to the formation of the network, the uptake of radioactive uridine occurred mainly around the granulated nuclear material. After the network had developed, the uptake of radioactive uridine was reduced, but some of the radioactivity appeared over the strands of the network (Fig. 13). Autoradiographs of infected cells treated with 3H-tyrosine during the course of granulosis indicated that the incorporation pattern and the amount of uptake of “H-tyro-’ sine were similar to those of 3H-uridine (Figs. 14-16). This suggested that protein synthesis in the infected cell during the course of the granulosis progressed in close association with RNA synthesis. Protein synthesis was pronounced in the whole nucleus up to the time of network formation, and thereafter the main part of the protein synthesis was restricted generally to the strands of network. Electron microscopy. At present the hypothesis is that the development of granulosis virus begins in the cell nucleus and continues, after an early rupture of the nuclear membrane, in both the nucleus and cytoplasmic areas (Huger and Krieg, 1961; Bird, 1963). Our electron microscope observations and autoradiographs presented herein support this hypothesis, i.e., that the granulosis virus is probably of nuclear origin. Electron micrographs of the infected fat body at a stage when the virogenic stroma exhibited a network revealed that a number
of virus rods appeared to protrude from strands of very dense material of the network into the spaces between the strands, where the rods were enveloped with a double membrane (Figs. 17 and 18). The mrclear membrane was completely destroyed by this time. Some long filaments of the‘ same thickness and density as virus rods were observed during the development of the virus ( Steinhaus et al., 1949; Smith aud Brown, 1965). Around the periphery of the strands, the virus particles were occluded in capsule protein and the capsules accumulated in the spaces between the strands. The occlusion of the virus particle by the capsule protein began mostly at one end of the virus rod as reported by Hughes (1952) and Bird (1964) and rarely began at both ends at the same time as observccl by Arnott and Smith (1968). The virus particles contained in the capsule were rod-shaped and varied from 5060 nq4 in diameter and from 280-350 rn!k in length. The capsules varied in size from 220-340 rnp in diameter and from 430600 rnp in length. The macromolecular lattice of the capsule protein was observed in thin sections. Double infection of granulosis and nucleopolyhedrosis viruses. Several insect species have been found infected simultaueouslv with both granulosis and nucleopolyhedrosis viruses (Tanada, 1953, 1956; Steinhaus, 1957; Bird, 1959). In H. cunea, mixed infection by granulosis and nucleopolyhedrosis viruses was obtained by feeding the larvae the mulberry leaves smeared with a mixed inoculum of the two viruses. The light microscope sections of the fat body with mixed virus infections showed
FIG. 17. Section through a fat-body cell of a larva of Hyplzat~tria cunea infected with a granulosis virus. Portion of virogenic stroma showing an abundance of capsules in spaces between the network strands and a number of virus rods scattered on the strands. X6,500. FIG. 18. Section through a fat-body cell of a larva of Hyphantria cuneu infected with a granulosis virus. Portion of virogenic stroma showing protrusion of virus rods from the strands of network and the development of the capsule around the virus rod. X 17,000.
HISTOPATHOLOGY
OY
A C;HA h Ii L,c his
.&i
78
WATANABE
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POBAYASHI
FIG. 19. Section through a fat body of a larva of Hyphtria curlea infected simultaneously with a nucleopolyhedrosis and a granulosis virus. The left cell is infected with a nucleopolyhedrosis virus and the right cell with a granulosis virus. Nucleopolyhedrosis virus ( NV), polyhedra (P), nuclear membrane (NM), cell membrane ((%I), granulosis virus rod (V), and capsule (C). X6,000.
that no one cell was infected simultaneoushby both viruses. This was aIso the case with the mixed virus infections in the larvae of Chorktoneura fumiferana and Trichoplusia ni according to Bird (1959) and Lowe and Paschke (1968). Although we have made numerous attempts with the electron microscope to find a cell with mixed virus infections, we were unsuccessful. However, WC’ did observe adjacent cells infected by different viruses (Fig. 19).
ACKWWLUXXENTS
Appreciation is expressed to Professor Y. Tanada of University of California for reviewing the manuscript and offering suggestions for its improvement.
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